An epithelial tissue, which is a cell layer covering the inside and outside surface of an animal body, such as an epidermis, a corneal epithelium, an alveolar epithelium, a mucosal epithelium of digestive system, renal glomerular epithelium, and hepatic parenchymal cells, prevents the invasion of an exogenous material (microorganism, allergen, chemical substance, etc.) from the external world. The outer interface of epithelial cells that constitutes said epithelial tissue is called apical surface, and the inside undersurface is called basal surface. Just beneath said basal surface, there is a thin film structure of 50-100 nm thickness called a basement membrane comprised of extracellular matrices (ECM) such as proteins, and proteoglycans, which does not include cells. A basement membrane is considered to be an essential structure for immature epithelial cells to proliferate, to differentiate into mature cells, and to express its intrinsic morphology and function. In other words, without a basement membrane, an epithelial tissue cannot maintain itself or achieve its intrinsic performance. Although an epithelial cell layer of multilayer or monolayer prevents the invasion of an exogenous material from the external world as a barrier, a basement membrane itself also acts as a physical barrier. Thus, epithelial cells constituting an epithelial tissue collaborate with a basement membrane to form a solid barrier and to protect the internal vital activity.
A basement membrane, which is a specific membranous structure of extracellular matrices formed on the interface between parenchymal cells, such as epithelial cells, endothelial cells, muscle cells, adipocytes, Schwann cells, and connective tissues, is universally found in respective tissue/organ of a living body, however, some kinds of basement membranes are highly specialized such as a renal glomerular capillary loop, a nervous synapse membrane. Thus, it is disclosed that the basement membrane has not only a function to allow cells to adhere to interstitium, but also a function of selective permeability about substance/cell, and to induce the differentiation of cells. In renal glomerulus, negative electric charges of a basement membrane are considered to be responsible for the filtration function of kidney, and said negative electric charges are traditionally known to be due to heparan sulfate proteoglycan (HSPG) which is currently called perlecan. HSPG is widely distributed not only to the renal glomerular basement membrane but also to various kinds of basement membranes as a basic component of basement membrane, just like type IV collagen, laminin, entactin (nidogen), etc.
An extracellular matrix, especially a basement membrane, is now gradually known to be deeply involved not only in the physiological phenomena such as generation and differentiation of an individual as described above, but also in the formation of pathology such as proliferative metastasis of cancer and inflammation. Therefore, clarification of the function of its constituent protein has been an important task. For example, laminin, which is a main glycoprotein of basement membrane, is a complex comprised of three subunits α, β, and γ, fifteen types of its isoforms are known, and they are expressed tissue-specifically, or specifically at each step of development. Laminin is a complicated macromolecule of 900,000 molecular weight having various bioactivities. As laminin receptors, integrin molecule such as α6β1, α-dystroglycan (α-DG), heparan sulfate proteoglycan (HSPG) of syndecan-1 to -4 are reported.
The interaction between a component of basement membrane, which is a thin extracellular matrix layer to which cells can adhere, and epithelial cells influences the cell function such as migration, proliferation, and differentiation (Crouch et al., Basement membrane. In The Lung (ed. R. G. Crystal and J. B. West), pp 53.1-53.23. Philadelphia: Lippincott-Raven. 1996). As for the main components of basement membrane, laminin, type IV collagen, heparan sulfate proteoglycan (HSPG), and entactin (nidogen) are known as described above (Curr. Opin. Cell Biol. 6, 674-681, 1994), and mesenchymal cells are considered to play an important role for the synthesis of basement membrane components including isoforms of laminin and type IV collagen (Matrix Biol. 14, 209-211, 1994; J. Biol. Chem. 268, 26033-26036, 1993), however, the role of epithelial cells is also important. HSPG has been believed to be derived from epithelial cells, however, laminin, type IV collagen, and entactin (nidogen) are synthesized in vivo by both of epithelial cells and mesenchymal cells (Development, 120, 2003-2014, 1994; Gastroenterology 102, 1835-1845, 1992). Many attempts have been made to construct an epithelial tissue model in vitro showing a continuous lamina densa. Tissue models of intestine (J. Cell Biol. 133, 417-430, 1996) and skin (J. Invest. Dermatol., 105, 597-601, 1995; J. Invest. Dermatol., 109, 527-533, 1997; Dev. Dynam., 197,255-267, 1993) and the like have been studied, and some of basement membrane components derived from mesenchymal cells have been found to play an important role in the formation of basement membrane.
Several methods to constitute a basement membrane by culturing epithelial cells, and to constitute epithelial tissues wherein a basement membrane structure is present just beneath the basal surface have been reported. For example, the present inventor has reported that a basement membrane can be formed in vitro by coculturing alveolar epithelial cells and pulmonary fibroblasts (Cell Struc. Func., 22:603-614, 1997). It has been reported that when pulmonary fibroblasts are embedded in type I collagen gel and cultured, the collagen gel is contracted and becomes more solid by pulmonary fibroblasts. Extracellular matrices secreted from pulmonary fibroblasts attach to collagen fibrils around the cells and are deposited; the resultant formation is called a pseudointerstitium since it is similar to an interstitium in vivo. Basement membrane components such as laminin, type IV collagen, perlecan, entactin (nidogen) are also secreted from pulmonary fibroblasts to the culture medium. When alveolar type II epithelial cell line (SV40-T2) was cultured on such pseudointerstitial type I collagen fibrils for approximately 14 days (T2-Fgel), basement membrane components secreted from pulmonary fibroblasts diffuse and reach the basal surface of the alveolar type II epithelial cell line described above, and are used as materials for the constitution of basement membrane, and as a result, a basement membrane structure is formed.
It is also reported that a basement membrane is formed just like the case of coculture of the above alveolar epithelial cells and pulmonary fibroblasts by the following process: the diluted neutral collagen solution was incubated at 37° C. in 5% CO2, and collagen fibrils were formed, then the air-dried fibrillar collagen matrix (fib) that was air-dried in aseptic condition was used as an alternative to the pseudointerstitium described above (Eur. J. Cell Biol., 78:867-875, 1999; J. Cell Sci., 113:859-868, 2000). In this process, if the concentration of the collagen solution is high, there will be less or no space formed among fibrillar collagen, and if epithelial cells are cultured for a long term (10 days-2 weeks) for the purpose of basement membrane formation, the cells are exfoliated and floated (e.g. Becton Dickinson, Fibrillar collagen coat culture insert), therefore, the concentration of the collagen solution is considered to be optimum at 0.3-0.5 mg/ml (Eur. J. Cell. Biol., 78:867-875, 1999; J. Cell Sci., 113:859-868, 2000).
Alveolar type II epithelial cell line (SV40-T2) was cultured on a fibrillar collagen matrix coexistent with Matrigel (the registered trademark of Becton Dickinson), instead of using a collagen matrix wherein fibroblasts were embedded. In this case, Matrigel functioned as an exogenous resource of basement membrane components. Matrigel is a basement membrane preparation extracted from Engelbreth-Holm-Swarm tumor matrix (J. Exp. Med. 145, 204-220, 1977), and contains laminin-1, entactin (nidogen), type IV collagen, and perlecan, as well as various kinds of cytokines that possibly influence the synthesis of ECM (Exp. Cell Res. 202, 1-8, 1992). In order to trace the incorporation of Matrigel-derived components into a basement membrane, Matrigel was labeled with biotin. Among the labeled basement membrane components, laminin-1 and entactin (nidogen) mostly diffused into the culture medium, and were incorporated in the basement membrane formed by pulmonary epithelial cells. In addition, when basement membrane components were stained immunofluorescently and observed with a (fluorescent) microscope, the acceleration of basement membrane formation depending on the amount of Matrigel, and the process wherein basement membrane matrix components are secreted, deposited in a punctiform manner, expanded in a sheet form, and then developed into a basement membrane were observed. Based on these results, it has been found that exogenous laminin-1 and entactin (nidogen) provided from a lower part below the basal surface of alveolar epithelial cells are largely involved in the in vitro formation of a complete basement membrane by the epithelial cells described above (J. Cell Sci., 113:859-868, 2000).
Further, as a method for culturing cells comprising the attachment of cells in vitro, a method wherein an end-group activated polymer (EGAP) conjugated with a biomolecule via disulfide bond is adsorbed to the surface of hydrophobic tissue culture, and cells are seeded on the surface of the biomolecule-conjugated EGAP coating and proliferated is disclosed (Published Japanese Translation of PCT International Publication No. 2001-512565; WO98/31734).
When cultured cells adhere to a substrate, receptor molecules, such as integrin, present on the cell surface are generally used for the adhesion. Therefore, in order to induce the cell adhesion to a substrate by using specific receptors, extracellular matrices, etc. that have cell adhesion activities are used as a linker. As for representative extracellular matrices, cell adhesion proteins such as fibronectin (FN), collagen (Col), laminin (LN) and vitronectin (VN) are known. These cell adhesion proteins are used as a substrate for cell culture. In the culturing process, these cell adhesion proteins are directly adsorbed by noncovalent binding by making use of hydrophobic binding to plastic wells, and epithelial cells, vascular endothelial cells, fibroblasts, etc., are seeded thereon. However, these cell adhesion proteins are expensive and, what is more, they are susceptible to denaturation and degradation, which are general properties of proteins, and have problems with its price, stability, shelf life, etc.
In addition, a peptide in the region relating to the cell adhesion in the amino acid sequence of a cell adhesion protein can be directly adsorbed to plastic wells in the manner similar to that described above and used as a substrate for cell culture. In comparison with a method using these cell adhesion proteins, a method using peptides which can be easily chemosynthesized as cell adhesion peptides has advantages when it is used for a cell culture substrate because such peptides are easily mass-produced and these structure are relatively stable. However, because such peptides have a low molecular weight, their adsorptive efficiency is extremely lower than that of proteins, and only a few percent of them are adsorbed. Further, it is difficult for the peptides as well to bind to receptors on the cell side when they are adsorbed to plastics and deprived of the freedom of movement. In addition, even if peptides once adsorbed to the plastics the peptides will be gradually detached from there subsequently. Therefore, the reproducibility of cell adhesion using peptides is not excellent, and it (the wells where peptides are directly coated) is not highly valuable as industrial goods.
The present inventor has found a method for coating the hydrophobic surface of a well with a cell adhesion ligand (a molecule of an extracellular matrix to which a receptor binds) to induce the adhesion of cultured cells to a substrate, instead of a hydrophilically treated plastic well used for cell culture, and completed the present invention. This method makes use of noncovalent bonding (hydrophobic binding) between a ligand, a receiver of cell adhesion, and a well on which the ligand is immobilized.
In other words, the present invention is to provide: a cell culture substrate which adsorbs to cell culture substrates such as wells, and is excellent in the reproducibility of cell adhesion, and whose surface is coated with a hydrophobic binding-adsorptive polymer; the immobilized preparation of cell adhesion proteins or peptides which binds to cell culture substrates efficiently, and is excellent in the reproducibility of cell adhesion; and further, an artificial tissue prepared by seeding cells onto the immobilized preparation of cell adhesion peptides and culturing the cells.